Validation of a Rapid Method for Measuring β-Glucosidase Activity in Fermenting Muscat Grape Musts

Abstract

Interest in β-glucosidases has focused upon their activity in different yeast strains and commercial enzyme products, for conversion of monoterpene glycosides to free volatile terpenes, thereby increasing wine aroma intensity. This research involved modifying a unique spectrophotometric assay based on β-glucosidase hydrolysis of <i>p</i>-nitrophenyl β-<i>O</i>-d-glucopyranoside (PNPG), resulting in a chromophore complex with α-cyclodextrin. The validity of the assay was tested and it was then used to measure β-glucosidase activity in muscat fermentations with various yeast strains or commercial enzyme additions. The assay quantified β-glucosidase activity following Michaelis–Menten-like kinetics using β-glucosidase from almond extract (K<i>m</i> of 7.15 mM PNPG). The kinetics of an enzyme isolated from <i>Saccharomyces cerevisiae</i> strain VL1 fermentation exhibited similar results (K<i>m</i> of 6.28 mM PNPG). An enzyme preparation (AR2000) demonstrated higher glucosidase activity than a control under various pH, temperature, and incubation times. The assay was then tested on VL1 yeast inoculated into muscat must; β-glucosidase activity peaked after 24 hr followed by a drop in activity. Greater increases in chromophore formation occurred after the first day isolates were allowed to incubate for 48 hr. Similar patterns of activity were observed when muscat must was inoculated by each of six yeast strains, whereby <i>S. bayanus</i> strain EC1118 and <i>S. cerevisiae</i> strain VL1 were highest in β-glucosidase activity. Wines produced with these yeasts tended to be highest in melon and muscat aromas.